Recent investigations have revealed the great potential of oligonucleotide and oligonucleotide analogues as chemotherapeutic agents (see P. Miller and P. Ts'o, Ann. Rep. Med. Chem. 23: 295, 1988; G. Zon, Pharm. Res. 5: 539, 1988). As reported by Matsukura and colleagues (Proc. Natl. Acad. Sci. USA 84: 7706, 1987), certain analogues of oligonucleotides whose normal backbone structure has been changed from phosphodiester to phosphorothioate linkages are capable of potent inhibition of the replication of the human immunodeficiency virus (HIV). More recently, Matsukura et al (Proc Natl Acad Sci USA 68: 4244, 1989) have shown that the expression of specific HIV genes can be regulated with phosphorothioate oligonucleotides. Also, the results of a F&DA approved Phase I clinical trial of systemic administration of OL(1)p53 (SEQUENCE ID NO: 1) in refractory acute myelogenous leukemia and advanced myelodysplastic syndrome are reported by P. L. Iversen et al (J Am Soc Hem., Nov. 15, 1993 Supplement, page 443a, abstract 1757).
Until discovered by the present inventor, however, it was not recognized that the binding of oligonucleotides, of various sizes, to the outer membrane surface of biologically living cells could lead to the production of deleterious mutational events in the cell targeted by the oligonucleotide. This finding prompted a search for potential mechanisms by which the binding of an oligonucleotide to a targeted cell could lead to mutational events. Subsequent experimentation led to the discovery that large amounts of reactive free hydroxy radicals (OH.sup.-) were released by the cell which bound the oligonucleotide. (See Patrick L. Iversen U.S. patent application Ser. No. 07/735,067, filed Jul. 25, 1991 entitled INHIBITION OF MUTAGENICITY INDUCED BY BINDING OF OLIGONUCLEOTIDES TO CELLS.)
Considerable evidence exists in the art that free oxygen radical species have highly mutagenic capabilities. The unexpected release of reactive oxygen radicals (especially free hydroxy radicals) by cells following the binding of oligonucleotides to the surface of those cells is in stark contrast to prior understanding of the biological consequences of binding of oligonucleotides to cell membranes.
Partially reduced forms of oxygen arise continually in all cells as products of normal metabolic pathways. Such highly reactive oxygen intermediates can be highly toxic, however, if they are not immediately inactivated or utilized in metabolic pathways. For example, in a cell, a common pathway for the complete reduction of one molecule of O.sub.2 is to form water in a four-electron transfer process. However, cellular metabolic pathways also continually generate incompletely reduced species of oxygen. A one-electron reduction of O.sub.2 yields superoxide ion, O.sub.2.sup.- ; an additional electron yields hydrogen peroxide, H.sub.2 O.sub.2 ; and a third electron yields a hydroxy radical, OH.sub.2.sup.-, and a hydroxide ion. These so-called "reactive oxygen radicals" are far more reactive, and hence potentially much more toxic, than is O.sub.2 itself.
Hydroxy radicals, in particular, are extremely reactive and represent the most active mutagen derived from ionizing radiation. The radical is highly electrophilic and reactive, with a capacity to bind to DNA to produce modified bases, such as 8-hydroxyguanine, and thymine glycol. The former has been detected in DNA isolated from tissues exposed to ionizing radiation or to hydrogen peroxide (H.sub.2 O.sub.2) (Kasai et al., Carcinogenesis 7: 1849, 1986). The latter, which is an oxidation product of thymine residues in DNA, is often found in the urine of individuals who have suffered DNA damage (B. Ames, Science 221: 1256-1264, 1983).
It is known in the art that hydroxylated derivatives of DNA are generated continuously by normal cellular metabolism, and that such modified DNA is most commonly repaired by unscheduled DNA synthesis. If, however, the modified DNA bases remain unrepaired, the consequences of the random DNA damage can include (i) mutagenesis due to erroneous replication of damaged DNA nucleotide base templates, and/or (ii) cell death due to inability of the cell to replicate its genome past a damaged DNA site. The generation of excessive amounts of reactive oxygen intermediates can, therefore, have serious ramifications for the host organism.
Most cells normally contain one or more enzyme systems which very rapidly combine with and inactivate excess reactive oxygen species. One of the major enzyme systems in this regard is the superoxide dismutase (SOD) family of metalloenzymes. Superoxide dismutase detoxifies two molecules of superoxide simultaneously, oxidizing one molecule while reducing the other: ##STR1## One form of this metalloenzyme is found in the cytoplasm of eukaryotic cells and contains copper and zinc; a different form is found both in mitochondria and in bacterial cells, and contains manganese; and another related iron-containing form is found in some bacteria, cyanobacteria, and some plants (see, for example, C. K. Matthews and K. E. van Holde, Biochemistry, The Benjamin/Cummings Publishing Company, Inc., Redwood City, Calif., 1990). The wide occurrence of SOD enzymes is confirmation of the biological necessity of rapid inactivation of reactive oxygen intermediates.
Hydrogen peroxide (H.sub.2 O.sub.2), another highly reactive oxygen species, is inactivated by at least two different enzyme systems. The most commonly utilized is the enzyme catalase, which is a heme protein widely distributed in cells. ##STR2## The reaction involves oxidation of one molecule of H.sub.2 O.sub.2 and reduction of another. The extremely high turnover rate of this enzyme (more than 40,000 molecules utilized per second) confirms the importance of removing excess hydrogen peroxide from the cellular micro-environment.
Another family of hydrogen peroxide scavengers is the peroxidases which reduce H.sub.2 O.sub.2 to water while simultaneously oxidizing an organic substrate. For example, erythrocytes contain a selenium-containing enzyme, glutathione peroxidase (GSH), which reduces H.sub.2 O.sub.2 to water while simultaneously oxidizing the glutathione: ##STR3## Glutathione peroxidase contains one residue per mole of an unusual amino acid, selenocysteine, an analog of cysteine that contains selenium in place of sulfur. Glutathione synthetase and glutathione reductase are further examples of this family of scavengers.
Lastly, it is known in the art that H.sub.2 O.sub.2 is chemically inactivated by mannitol, with which it forms a stable, equimolar compound. For the purposes of this invention hydroxy, H.sub.2 O.sub.2, and superoxide ion are considered to be interchangeable.
Other chemical constituents which are well known in the art for their anti-oxidant potential are such chemicals as, for example, vitamin A (Retinol and retinoic acid); vitamin E (alpha-tocopherol); Vitamin C (ascorbic acid); and the trace mineral element selenium, all of which serve in a variety of tissues and bodily processes as general reducing agents.
Cancer cells, particularly those which are malignant, exhibit elevated levels of free hydroxy radicals. These and other types of diseased cells do not exhibit the same degree of anti-oxidant protection as do normal cells. In particular, such cells are notably deficient in the scavenger protection systems discussed above. Because of this fact, irradiation kills cancer cells preferentially to normal tissue and conventional radiation therapy attempts to exploit this mechanism of action, as do certain conventional chemotherapeutic agents, such as the nitrosoureas, e.g., BCNU (bis-chloroethylnitrosourea), and the anthracycline cytotoxic antibiotics, doxorubicin and daunorubicin.
However, the sensitivity of a cell and the resultant cellular response to ionizing radiation depends primarily on the presence or absence of oxygen within the cell and upon the stage of division which the cell is in at the time of irradiation. In radiation therapy, production of oxidative damage is initiated by a dose of radiation. Cells which are oxygen-rich and sensitive to radiation will be killed more effectively and efficiently than cells which are oxygen-deficient. It appears that the molecular pathways responsible for inherent radiation sensitivity involve the initiation and production of oxidative damage, cellular sensing of the damage, and cellular response to and repair of the damage. Repair of radiation damage is most likely an enzymatic process and radiobiologists have examined the role of certain enzymes which are responsible for the detoxification of cytotoxic oxygen-related free radicals. (Golfman T. E., et. al., Cancer Research, 50: 7735-7744, 1990)
Within the cell cycle is an arrest stage, which allows the cell to stop its growth mechanism for a period of time long enough to repair any DNA damage which is detected. In the repair process, segments of DNA which contain an altered base can be recognized and repaired. When an irradiated mutant (i.e. cancerous) cell is given time to repair, the mutated DNA will be allowed to continue in its division and replication, thus proliferating the cancer or tumor.
The cellular repair mechanism is located between the G.sub.1 and S phase of the cell cycle. The gene p53 has been postulated to play a role in the repair of damaged DNA and is in fact considered to function as a cell cycle checkpoint after irradiation. (Lee, J. M., and Bernstein A, Proc. Nat. Acad. Sci., 90(12): 5742-5746, 1993). Apoptosis is part of normal development and also can be triggered by DNA damage, such as that delivered by radiation and some chemicals, including those used in chemotherapy. It has been shown that levels of p53 protein rise dramatically after DNA damage. Consequently, p53 is currently believed to be crucial to the apoptotic pathway induced by DNA damage.